Ozer R S, Halpain S
Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Mol Biol Cell. 2000 Oct;11(10):3573-87. doi: 10.1091/mbc.11.10.3573.
Microtubule-associated protein 2 (MAP2) is a neuronal phosphoprotein that promotes net microtubule growth and actin cross-linking and bundling in vitro. Little is known about MAP2 regulation or its interaction with the cytoskeleton in vivo. Here we investigate the in vivo function of three specific sites of phosphorylation on MAP2. cAMP-dependent protein kinase activity disrupts the MAP2-microtubule interaction in living HeLa cells and promotes MAP2c localization to peripheral membrane ruffles enriched in actin. cAMP-dependent protein kinase phosphorylates serines within three KXGS motifs, one within each tubulin-binding repeat. These highly conserved motifs are also found in homologous proteins tau and MAP4. Phosphorylation at two of these sites was detected in brain tissue. Constitutive phosphorylation at these sites was mimicked by single, double, and triple mutations to glutamic acid. Biochemical and microscopy-based assays indicated that mutation of a single residue was adequate to disrupt the MAP2-microtubule interaction in HeLa cells. Double or triple point mutation promoted MAP2c localization to the actin cytoskeleton. Specific association between MAP2c and the actin cytoskeleton was demonstrated by retention of MAP2c-actin colocalization after detergent extraction. Specific phosphorylation states may enhance the interaction of MAP2 with the actin cytoskeleton, thereby providing a regulated mechanism for MAP2 function within distinct cytoskeletal domains.
微管相关蛋白2（MAP2）是一种神经元磷蛋白，在体外可促进微管的净生长以及肌动蛋白的交联和束集。关于MAP2在体内的调控或其与细胞骨架的相互作用，目前所知甚少。在此，我们研究了MAP2上三个特定磷酸化位点的体内功能。环磷酸腺苷（cAMP）依赖性蛋白激酶活性破坏了活的HeLa细胞中MAP2与微管的相互作用，并促进MAP2c定位于富含肌动蛋白的外周膜皱褶。cAMP依赖性蛋白激酶使三个KXGS基序内的丝氨酸磷酸化，每个微管蛋白结合重复序列中有一个。在同源蛋白tau和MAP4中也发现了这些高度保守的基序。在脑组织中检测到其中两个位点的磷酸化。通过将单个、双个和三个位点突变为谷氨酸来模拟这些位点的组成型磷酸化。基于生化和显微镜的分析表明，单个残基的突变足以破坏HeLa细胞中MAP2与微管的相互作用。双点突变或三点突变促进MAP2c定位于肌动蛋白细胞骨架。去污剂提取后MAP2c与肌动蛋白的共定位得以保留，证明了MAP2c与肌动蛋白细胞骨架之间的特异性结合。特定的磷酸化状态可能增强MAP2与肌动蛋白细胞骨架的相互作用，从而为MAP2在不同细胞骨架结构域内的功能提供一种调控机制。
